Competition between stochastic neuropeptide signals calibrates the rate of satiation

We investigated how transmission of hunger- and satiety-promoting neuropeptides, NPY and αMSH, is integrated at the level of intracellular signaling to control feeding. Receptors for these peptides use the second messenger cAMP. How cAMP integrates opposing peptide signals to regulate energy balance, and the in vivo spatiotemporal dynamics of endogenous peptidergic signaling, remain largely unknown. We show that AgRP axon stimulation in the paraventricular hypothalamus evokes probabilistic NPY release that triggers stochastic cAMP decrements in downstream MC4R-expressing neurons (PVHMC4R). Meanwhile, POMC axon stimulation triggers stochastic, αMSH-dependent cAMP increments. Release of either peptide impacts a ~100 μm diameter region, and when these peptide signals overlap, they compete to control cAMP. The competition is reflected by hunger-state-dependent differences in the amplitude and persistence of cAMP transients: hunger peptides are more efficacious in the fasted state, satiety peptides in the fed state. Feeding resolves the competition by simultaneously elevating αMSH release and suppressing NPY release, thereby sustaining elevated cAMP in PVHMC4R neurons. In turn, cAMP potentiates feeding-related excitatory inputs and promotes satiation across minutes. Our findings highlight how biochemical integration of opposing, quantal peptide signals during energy intake orchestrates a gradual transition between stable states of hunger and satiety.


Introduction 32
Arcuate hypothalamic neurons integrate hormonal 1-4 , interoceptive 5-7 , and external cues [8][9][10][11][12] and 33 use neuropeptides to regulate food intake, energy expenditure, and body weight [13][14][15][16][17][18] . In particular, 34 AgRP neurons release hunger-promoting agouti-related peptide (AgRP) and neuropeptide Y 35 (NPY) 19-24 , while POMC neurons release the satiety-promoting α-Melanocyte-stimulating 36 hormone (αMSH) among other peptides 13,25-28 ( Figure 1A). These peptides bind G-protein 37 coupled receptors (GPCRs) that signal by increasing 29 or decreasing 30 cAMP concentration. 38 Mutations in these receptor-to-cAMP cascades are associated with obesity in both humans and 39 experimental models 31-40 . One important target of these peptides is the satiety-promoting MC4R-40 expressing neurons in the paraventricular nucleus of the hypothalamus (PVH MC4R ) 4,13,14,24,41-46 . 41 How these hunger and satiety peptides are integrated at the level of cAMP, and how cAMP, in 42 turn, controls spiking of PVH MC4R neurons remain largely unknown. One major obstacle has been 43 the inability to directly monitor cAMP levels in individual PVH MC4R neurons in vivo, because cAMP 44 dynamics are invisible using conventional electrophysiological or calcium recordings. In the 45 absence of direct cAMP measurements, past studieswhich have largely relied on indirect 46 measurements and pharmacologyhave generated conflicting results regarding the involvement 47 of cAMP in regulating PVH MC4R neuron activity and hunger 47-54 . There is a critical need to assess 48 neuropeptide release and intracellular signaling dynamics in the awake brain, where neurons are 49 surrounded by many competing peptides whose concentrations vary across behavioral states. 50 To overcome these technical challenges, we have recently assembled a suite of tools to stimulate 51 and track endogenous neuropeptide release from AgRP and POMC neurons, as well as to 52 measure and manipulate cAMP levels in individual PVH MC4R neurons, in awake mice across 53 seconds to hours 55 . Neuropeptides are found in dense-core vesicles that sparsely populate 54 chemical synapses 56 . Once released, these neuropeptides could potentially diffuse across long 55 distances to signal downstream neurons 56-59 . However, many questions regarding this process 56 all trials (Figure 2H) or only hit trials (Figure 2I). POMC axon-evoked cAMP increments showed 152 ~80% larger peak amplitudes (Figure 2J) and longer durations (>5 min vs ~100 s; Figure S2B) in 153 the fed state than in the fasted state. Accordingly, persistence (calculated as the ratio of the mean 154 cAMP response magnitudes from 20-40 s and from 80-100 s after stimulus onset) was five-fold 155 greater in fed mice (Figure 2K). Together, these results demonstrate satiety state-dependent 156 enhancement in the efficacy of second messenger signaling downstream of POMC axon 157 activation. 158 Although the above results are consistent with αMSH-MC4R mediated cAMP increments (Figure 159

S3B). 187
Out of all the neuropeptides and small-molecule neurotransmitters that AgRP neurons could 188 release (e.g., GABA, AgRP, NPY), NPY mediates a significant proportion of the hunger-promoting 189 effects, especially the slow effects that last many minutes 20 . Therefore, we tested whether NPY 190 is the main mediator of AgRP-to-PVH MC4R  AgRP or POMC stimulation were state-dependent. Below, we investigated the mechanisms of 203 stochasticity in cAMP signaling (Figure 4), the causes of state-dependent differences in 204 persistence of cAMP transients (Figure 5), and the implications of cAMP stochasticity and 205 persistence for PVH MC4R neuron activity and feeding (Figures 6-7). 206 207 A stochastic and spatially restricted mode of peptide release 208 The probabilistic nature of the neuropeptide-mediated cAMP signaling described above is 209 redolent of stochastic neurotransmitter release 86 , albeit with the major distinction that the 210 probability of a cAMP transient is low despite bulk stimulation of many AgRP or POMC axons. 211 This low probability is consistent with ultrastructural data showing that most AgRP and POMC 212 presynaptic boutons harbor only a few peptide-containing dense-core vesicles that are sparsely 213 distributed among hundreds of small clear vesicles 73 . To ascertain whether stochasticity in cAMP 214 signaling reflects stochasticity in neuropeptide release, we used viral expression of a new 215 fluorescent NPY sensor, npyLight, in PVH neurons ( Figures 4A and S4A). We first confirmed this 216 sensor was sensitive to 200 nM NPY in brain slices (Figure 4B). To induce endogenous NPY 217 release, we used trains of Chrimson photostimulation (15.5 Hz) of varying duration (2, 4, 8, 16 s) 218 to activate AgRP axons in the PVH in brain slices ( Figures S4B-S4E). Similar to the stochastic 219 nature of cAMP decrements, AgRP axon photostimulation only triggered npyLight transients in a 220 subset of trials ( Figures S4B-S4E). These npyLight transients were mostly well described by the 221 dice model, with an estimated release probability of ~15% that increased only slightly with 222 stimulation duration ( Figures 4C-4D and S4F). Moreover, the amplitude of hits remained 223 consistent for all stimulation durations ( Figures 4E-4F). Due to the lack of an αMSH sensor, we 224 could not test these hypotheses in the POMC-to-PVH MC4R cAMP signaling pathway. Nevertheless, 225 these results are consistent with a model in which stochastic peptide release underlies the 226 unpredictability of peptide-mediated cAMP signaling in PVH MC4R neurons. 227 Whereas fast neurotransmission is mostly restricted to postsynaptic regions proximal to synapses, 228 neuropeptides can diffuse across longer distances 56-58 , sometimes estimated to span entire brain 229 areas 56,87-89 . A recent study using an oxytocin sensor indicated a spatial spread of ~100 μm in 230 hypothalamic slices 90 , demonstrating a local-diffusion mode of signaling 56 . Similar analyses have 231 not been done for αMSH and NPY, or for any peptide in vivo, so we set out to use our imaging 232 system to characterize the spatial impact and subcellular compartmentalization of NPY and αMSH 233

signaling. 234
We considered three potential modes of peptide transmission. First, in the discrete model, somas 235 and associated neurites function as independent peptide sensing compartments 91-95 , and show 236 largely uncorrelated cAMP signaling ( Figure 4G; although most neurite regions-of-interest (ROIs) 237 connected to a soma are likely dendrites, we termed these neurites to be conservative). Second, 238 in the large-volume transmission model 56,87,88 , neuropeptide signals are broadcasted widely 239 across somas and neurites, so cAMP signals should be correlated within the same cell and across 240 different cells ( Figure 4G). Third, in the cAMP diffusion model, neuropeptide transmission is locally 241 restricted but the resulting cAMP changes are seen in other compartments of the same cell due 242 to intracellular diffusion ( Figure 4G). In this case, trial-by-trial cAMP signals in somas and 243 associated neurites should be correlated. 244 We used the above logic to re-analyze the cAMP- (Figures 2-3) and NPY-imaging datasets 245 ( Figures 4A-4F). Initial support for the discrete model comes from single-trial analyses of a 246 PVH MC4R neuron soma and two connected neurites, all three of which show local cAMP 247 increments but on separate trials ( Figure S4G). To systematically characterize cAMP signals in 248 soma-neurite pairs of ROIs, we trained Cellpose 2.0 96 to automatically segment neurite ROIs, 249 followed by manual matching of neurites with associated somas (Figure S4H and Methods). 250 Analyses of AgRP-to-PVH MC4R cAMP imaging data indicated stochastic cAMP decrements in 251 neurites similar to those observed in somas ( Figures 3E and 4H). Further, neurite cAMP 252 decrements were more persistent in the fasted state, similar to soma decrements (Figures 3F-3G  253 and 4I). We next evaluated the presence or absence of concurrence between soma hits and 254 neurite hits. The neurite cAMP signals averaged across soma hit trials were weak and similar to 255 signals averaged across soma miss trials, indicating a lack of strong correlation between a cAMP 256 decrement in a neuron's soma and in its neurite ( Figure 4J). Likewise, neurite analysis of POMC-257 to-PVH MC4R cAMP imaging data also showed stochastic cAMP increments that were more 258 persistent in the fed state ( Figures 4K-4L). Changes in neurite cAMP were weak and of similar 259 amplitude when averaged across soma hit trials or miss trials, again indicating decorrelation of 260 soma and neurite signals ( Figure 4M). These results show that NPY and αMSH transmission to 261 PVH MC4R neurites largely follow the same rules as soma signaling, albeit with a lower hit probability 262 per surface area in neurites ( Figure S4H). However, the stochastic hits and misses are 263 decorrelated between somas and neurites of the same cell, therefore favoring the discrete 264 compartmental model over large-volume transmission or cAMP diffusion models. 265 The above correlation metric treats all soma-neurite pairs equally regardless of distance, which 266 may obscure correlations between close-range soma-neurite pairs. Such close-range correlations 267 may occur if a neuropeptide release event has a spatially restricted impact on adjacent but not 268 more distant targets ( Figure S4I). We used XNOR ( Figure S4J) as a similarity metric that 269 measures the coincidence of simultaneous hits or misses in soma-neurite pairs. We found that 270 for both AgRP-to-PVH MC4R and POMC-to-PVH MC4R transmission, concurrent cAMP changes in 271 soma-neurite pairs were much more common for pairs less than ~100 μm apart ( Figure S4K; 272 similar findings were also observed using other similarity metrics, Figures S4L-S4M). A similar 273 falloff of correlated cAMP signals with distance was also seen for recordings from somas of two 274 different cells and for NPY signals measured at different locations ( Figure 4N), demonstrating that 275 spatially adjacent somas can be impacted by the same plume of peptide release. These data 276 further support the claim that spatially localized peptide release-but not intracellular diffusion of 277 cAMP-defines the spatial scale of correlations in cAMP. Taken together, these results support 278 a discretized mode of neuropeptide signaling in which stochastic neuropeptide release events 279 evoke local cAMP changes in compartments within and across PVH MC4R neurons ( Figure 4O). enhanced during different hunger states, it is unlikely that a global state such as arousal dictates 286 the overall magnitude and persistence of peptidergic signaling ( Figure 5A). We tested whether 287 these hunger-state-dependent differences in signaling could be due to changes in i) axon 288 excitability, ii) cAMP clearance, and/or iii) cAMP production across fasted and fed states. To test 289 the first hypothesis, we asked whether photostimulation-evoked calcium transients in AgRP or 290 POMC axons differed across hunger states. Photometry measurements of Axon-GCaMP6s 97 in 291 vivo showed that our stimulation protocol triggers short-lived calcium transients with similar 292 amplitudes and decay kinetics in fasted and fed mice ( Figures S5A-S5B), ruling out the axon-293 excitability hypothesis. To test the second hypothesis, we compared the clearance kinetics of 294 cAMP produced by the blue-light-activated adenylyl cyclase, biPAC 55,98,99 . In brain slices from 295 fasted and fed mice, cAMP clearance in PVH MC4R neurons occurred within several minutes and 296 did not differ between fasted and fed states ( Figure S5C), ruling out the cAMP-clearance 297

hypothesis. 298
We then tested the hypothesis that cAMP production in PVH MC4R neurons was state dependent. 299 AgRP neurons are known to exhibit elevated ongoing activity in the fasted state 8-10,100-102 , which 300 likely results in a tonic level of NPY release that could hinder αMSH-induced cAMP production 301 ( Figure 5A). Likewise, in the fed state, steady-state levels of αMSH due to elevated tonic firing of 302 POMC neurons 8,9 could activate MC4R receptors to counter the suppression of cAMP production 303 by NPY ( Figure 5A). 304 To test this peptide competition hypothesis, we sought to artificially mimic this receptor 305 competition by introducing neuropeptide agonists that are known to induce artificial states of 306 hunger or satiety in behavioral experiments 42,80,81,103,104 ( Figure 5A). First, we tested whether 307 mimicking the tonic elevation in αMSH that likely occurs in fed mice is sufficient to attenuate, in 308 fasted mice, the amplitude and persistence of AgRP stimulation-induced cAMP decrements. As 309 expected, intraperitoneal (i.p.) pre-injection of melanotan II (MTII, 3 mg/kg), a satiety-promoting 310 MC4R/MC3R agonist ( Figure 5B), elevated cAMP levels in PVH MC4R neurons in fasted mice 311 ( Figure S5D). Although this manipulation did not affect the hit rate of AgRP stimulation-induced 312 cAMP decrements (Figures S5E), it reduced cAMP decrement amplitude by 46% and persistence 313 by 41% ( Figure 5C) in a manner similar to our observations in fed mice ( Figure 3G). To more 314 directly simulate the effects of persistently elevated αMSH, we replaced the i.p. MTII delivery with 315 i.c.v. pre-infusion of 1 nmol of αMSH 103 (2 µl), a level of αMSH that was sufficient to elevate cAMP 316 in PVH MC4R neurons ( Figures S2E-S2G). αMSH delivery caused an even greater reduction in the 317 amplitude (78%) and persistence (86%) of AgRP stimulation-evoked cAMP decrements in the 318 fasted state (Figures 5D-5E and S5F-S5G). We also tested if NPY delivery 80,81,104 (0.5 nmol in 1 319 µl, i.c.v.) would weaken POMC stimulation-evoked cAMP increments in fed mice ( Figure 5F). 320 Consistent with the peptide competition hypothesis, i.c.v. pre-infusion of NPY in fed mice (Figures 321 S3E-S3F) reduced cAMP increment amplitude by 54% and persistence by 83% ( Figures 5G and  322 S5H-S5I) in a manner similar to that observed in fasted mice (Figure 2I). While an MC4R 323 antagonist also reduced cAMP increment amplitude, it did not attenuate persistence ( Figure 2N), 324 demonstrating that merely blocking POMC-evoked signaling at MC4Rs without introducing 325 downstream competition at the level of cAMP is not sufficient to fully mimic state-dependent 326 modulation. 327 The above experiments demonstrate that elevated levels of hunger or satiety peptides mutually 328 blunt each other's cAMP signaling magnitude and persistence. According to this competition 329 model, brief elevation of a peptide due to prior axon activation should also locally enhance cAMP 330 signaling upon additional release of the same peptide. To test this idea, we identified rare cases 331 in which the same PVH MC4R neuron exhibited cAMP transients on two consecutive trials (likely 332 due to two peptide release events in the same spatial vicinity; Figure S5J). As predicted, cAMP 333 transients were strengthened on the second hit as compared to the first during both AgRP and 334 POMC stimulations ( Figures S5J-S5O). Likewise, concentrating the stimulations of peptide 335 release in time by shortening the inter-stimulation interval (from 52 s to 2 s) triggered cAMP 336 increments and decrements that lasted more than 10 min ( Figures S5P-S5Q). Together, these 337 results show that neuropeptide competition is dose-dependent. 338 339 Stochastic, persistent cAMP signaling during feeding defines the rate of satiation 340 The above findings regarding the peptide competition hypothesis indicate that cAMP levels in 341 PVH MC4R neurons are most effectively determined by the relative firing rates of AgRP axons as 342 compared to POMC axons. Such a system should be able to filter out 105 co-excitation or co-343 inhibition of AgRP and POMC neurons 7,9 , and be most effective in modifying cAMP levels when 344 the activity of AgRP and POMC neurons concurrently shifts in opposite directions ( Figure 6A). 345 Such antiphasic modulation of AgRP and POMC neuron activity has been observed for virtually 346 all regulators of energy balance (leptin 3,106 , ghrelin 8,107 , feeding 8,9 , exercise 108 ). Accordingly, i.p. 347 injection of the hunger hormone ghrelin, which stimulates AgRP neurons and inhibits POMC 348 neurons 8,107 , readily produced a strong and sustained cAMP decrement in PVH MC4R neurons 349 ( Figures S6A-S6B). 350 Feeding rapidly suppresses the firing of AgRP neurons and increases the firing of POMC 351 neurons 8,9 . For fasted mice that have learned that a cue will predict a food reward, the bulk of 352 these changes in neural activity occurs within seconds of food cue presentation and onset of food 353 consumption 8-11 . Consequently, early in a feeding bout, the rise of αMSH release from POMC 354 neurons should trigger cAMP increments in PVH MC4R neurons, while the decrease in AgRP activity 355 should withdraw competition and render the POMC-evoked cAMP increments persistent (Figure 356 6A). To experimentally characterize cAMP dynamics during natural feeding and test whether 357 stochastic cAMP signaling also occurs in this context, we designed a feeding task in which drops 358 of milkshake (Ensure; 15 µl/trial) were delivered to a head-fixed mouse with the same 60-s ITI as 359 the above optogenetic experiments. After training, mice rapidly consume each milkshake delivery, 360 albeit with higher lick rates in the fasted state than in the fed state 11 ( Figure 6B). 2p-FLIM 361 measurements of absolute cAMP levels show that, in both fasted and fed states, the average 362 cAMP level of PVH MC4R neurons gradually rises in the first ~4 trials and plateaus for the remainder 363 of the session while mice are still feeding ( Figures 6C and S6C). Even prior to food consumption, 364 cAMP levels in the fed state were higher than in the fasted state ( Figure 6C), consistent with 365 elevated baseline extracellular levels of satiety peptides. These results show that the majority of 366 elevations in cAMP occur early during feeding, when the opposing changes in AgRP and POMC 367 neuron activity are largest 8-11 . We therefore divided each session into a climbing epoch (Trials 1-368 4 of the session) and a steady-state epoch (Trials 5-15) when analyzing single-trial cAMP 369 transients ( Figures 6D-6E). We found that feeding-evoked cAMP transients are also stochastic 370 ( Figures 6D-6G), indicating that stochastic neuropeptide signaling is not merely an artifact of 371 optogenetic stimulation. As predicted by the drop in AgRP activity that coincides with the increase 372 in POMC activity during feeding ( Figure 6A; likely resulting in decreased competition from NPY), 373 single-trial feeding-related cAMP increments were persistent (Figures 6F-6G and S6D-S6E). After 374 the first four trials, both the rate and amplitude of hits stabilized at lower levels ( Figures 6D-6G), 375 resulting in the observed plateauing of intracellular cAMP concentration ( Figure 6C). 376 Is the simultaneous drop in AgRP activity and rise in POMC activity important for feeding 377 regulation? To answer this question, we bred POMC-Dre;AgRP-Cre mice 46 and independently 378 stimulated POMC neurons (with Dre-dependent ChR2) and inhibited AgRP neurons (with Cre-379 dependent hM4Di 21,109 ) ( Figure 6H). Either manipulation alone reduced feeding (see also 22,46 ), but 380 the combination of the two resulted in greater feeding suppression than either alone ( Figure 6H). 381 Together with the cAMP imaging data above, these results argue that peptide competition at the 382 level of cAMP production functions as a biochemical filter that favors anti-phasic changes in 383 hunger and satiety peptide levels. 384 385 Elevating cAMP in PVH MC4R gradually accelerates satiation 386 The above results strongly suggest that elevating cAMP in PVH MC4R neurons should be sufficient 387 to promote satiety. As an initial test, we used biPAC to bypass neuropeptide signaling and directly 388 stimulate cAMP production in PVH MC4R neurons. We found that a tonic, low level of biPAC 389 activation reduced feeding in a refeeding paradigm involving ad libitum access to milkshake 390 ( Figure 6I). To understand the timescale of the satiety-promoting effect, we designed a task in 391 which mice lick during an audible cue ("Tone") to obtain milkshake ("Reward") ( Figure 6J). The 392 purpose of the cue is to synchronize consumption and allow for trial-locked optogenetic 393 stimulation of cAMP production in some trials. We used one of two types of sessions on alternating 394 days, with each session containing 50 trials at 1 trial/min. In control sessions, no biPAC stimulation 395 was delivered. In experimental sessions, biPAC stimulation was delivered at random on 50% of 396 trials and consisted of 1 s of stimulation starting 5 s before cue onset ( Figure 6K). This randomized 397 biPAC stimulation design allows for within-session behavioral analysis, and the 5-s delay between 398 stimulation and cue onset allows for cAMP to reach peak levels during cue and reward delivery 399 (see Figure S5C). An example session is shown in Figure S6F. Over the course of 50 trials, lick 400 rates during cue and reward windows and success rates declined faster in the experimental 401 sessions involving biPAC stimulation ( Figures 6K and S6G-S6H). The lick rates and success rates 402 did not recover after an additional 10 min with no biPAC stimulation, indicating satiety rather than 403 transient disengagement. Furthermore, within an experimental session, we could not detect any 404 differences in behavioral responses between biPAC-stimulation trials and interleaved no-405 stimulation trials ( Figures 6L and S6I), showing that elevated cAMP drives a gradual 406 enhancement of satiety across many minutes. The drops in tone-and reward-evoked lick rates 407 over a given ten-trial period scaled with the number of biPAC stimulations during this period 408 ( Figures 6M and S6J), arguing that repeated elevations in cAMP have an accumulating, dose-409 dependent effect that gradually accelerates satiation. 410 cAMP sensitizes PVH MC4R neurons to feeding-related excitatory inputs 411 How cAMP in PVH MC4R neurons regulates feeding ultimately depends on how this second 412 messenger alters the activity of these satiety-promoting neurons. A previous study found that 413 prolonged incubation with αMSH in brain slices strengthens excitatory inputs to PVH MC4R 414 neurons 110 , but did not determine the involvement of cAMP or the dynamics of the synaptic 415 plasticity in vivo during feeding. 416 To address these questions, we used fiber photometry to record calcium transients in PVH MC4R 417 neurons with the sensor RCaMP1a 111 during the same conditioned feeding task as above, in 418 which fasted mice lick during an audible cue to obtain milkshake reward at one trial/min (see 419  us to hypothesize that cAMP regulates the rate at which the calcium response to feeding develops. 431 To test this, in a separate session we artificially drove cAMP production in these same PVH MC4R 432 neurons via brief biPAC photostimulation 5 s before each cue. Although the shape of each 433 PVH MC4R response to food consumption did not change, responses emerged earlier in the session, of dense-core vesicles at a rate of ~1 μm/s 121faster than typical axonal transport but perhaps 485 still insufficient to compensate for frequent dense-core vesicle release (which typically occurs 486 within a second 90 ). These predictions can be tested using novel sensors of dense-core vesicle 487 trafficking and fusion 119,122 . 488 In addition to preventing neuropeptide depletion, the sparsity of αMSH and NPY release, together 489 with their intrinsically persistent impact on cAMP, enables a high-resolution mode of information 490 accumulation during repeated food consumption events and associated changes in AgRP and 491 POMC neuron activity ( Figure 6). During feeding, the rapid changes in firing of AgRP and POMC 492 neurons likely reflect the amount of additional calories that will be gained by imminent feeding 493 behaviors 8,13,110 . The value of gradual cAMP accumulation is that it can integrate this estimated 494 calorie-intake information across each bout of food consumption to properly calibrate the 495 strengthening of excitatory inputs to PVH MC4R neurons and the associated rate of satiation with 496 calorie intake. This surprisingly slow mode of information integration via minutes-long biochemical 497 signalingeven following a single taste of foodmay underlie the effectiveness of slower meal 498 consumption in earlier meal termination and reduced weight gain 123-125 . Due to this slow and 499 integrative nature of biochemical signaling, it will be important to consider the activity of AgRP We hypothesize that the degree of elevation in cAMP determines the rate of satiation. If we 505 compare satiation rate to the speed of a vehicle, αMSH release from POMC axons and NPY 506 release from AgRP axons may be analogous to gas and brake pedals, respectively. Feeding 507 simultaneously steps on the gas pedal (i.e., increases POMC activity and αMSH release) and 508 releases the brake (i.e., inhibits AgRP activity and NPY release). An elevation in cAMP does not 509 immediately drive a switch to a sated state, but instead increases the speed of approach towards 510 satiety. This analogy also illustrates that the difference between the activity of POMC and AgRP 511 neurons is particularly important for understanding when and how these inputs to PVH MC4R 512 neurons will modify cAMP signaling. As such, concurrent increases or decreases in the activity of 513 both AgRP and POMC neurons (e.g., during injection of cocaine, amphetamine, and nicotine 7 ) 514 may not predict changes in feeding. Indeed, our findings show that cAMP signaling in PVH MC4R 515 neurons is particularly sensitive to opposite-direction changes in extracellular αMSH and NPY, 516 such as during feeding. Accordingly, MC4R agonists (e.g., setmelanotide 127 ) may be more 517 effective in promoting weight loss if used in combination with NPY antagonists or PDE inhibitors. 518 Hunger and satiety peptides inhibit each other's signaling efficacy (Figure 5). At a molecular level, 519 competition may take place through allosteric competition between the Gαs and Gαi binding sites 520 on an adenylyl cyclase protein 128-131 . This mutual inhibition effectively mitigates the impact of 521 fleeting changes in AgRP and POMC neuron activity (e.g., when a mouse finds an unexpectedly 522 inaccessible food source 8 ) on the progression towards satiety. Such friction may be well suited 523 for maintaining a bi-stable hunger-satiety dichotomy and slows down transition from low to high 524 cAMP during feeding, in addition to other mechanisms hypothesized to carry out similar functions 525 in this circuit 100,101,132-134 . 526 We inferred the presence of baseline levels of peptides from the experiments using agonists 527 Consistent with the idea that NPY transmission is responsible for the persistent hunger-promoting 542 effects observed tens of minutes after termination of sustained AgRP stimulation 20,137 , we show 543 that a grouped series of AgRP axon stimulations over 98 s results in a persistent decrease in 544 cAMP that outlasts the stimulation by many minutes (Figures S5P-S5Q). This may be due to an 545 accumulation of high levels of extracellular NPY that exceeds rates of clearance and breakdown. The peptide competition model predicts that reducing AgRP neuron activity in the fasted state, 557 when αMSH levels are low, is insufficient on its own to elevate cAMP levels in PVH MC4R neurons. 558 Early in the feeding assay, when AgRP neurons should be inhibited, we also did not observe 559 feeding-related calcium responses in PVH MC4R neurons that might be expected due to removal of 560 GABAergic inhibition or via potential closing of GIRK channels upon removal of NPY 44,140,141 . This 561 lack of effective disinhibition may reflect a lack of fast excitatory transmission onto PVH MC4R 562 neurons in fasted mice. These findings could explain why mimicking the suppression of AgRP 563 neurons alone causes only a moderate suppression of feeding ( Figure 6H and Krashes et al. 22 ) 564 as compared to the combined inhibition of AgRP neurons and excitation of POMC neurons that 565 mimics natural activity patterns during feeding 8,9 and that produces the largest satiety effects 566 ( Figure 6H; recently, the Brüning lab independently arrived at the same conclusion, personal 567 The 100-µm spatial impact of neuropeptide signaling 570 While it is proposed that most MC4Rs are found in the primary cilium near the PVH MC4R soma 142-571 144 , POMC synapses are most commonly found on the distal dendrites of PVH neurons 73 . The 572 ~100 μm impact diameter during stochastic release of feeding-related neuropeptides ( Figure 4N) 573 may resolve this discrepancy due to the spread of peptidergic signals throughout much of the 574 somatodendritic span of a given neuron. We note that this spatial scale is consistent with recent 575 studies of neuropeptide diffusion 58,90 , suggesting that local diffusion may be a general mode of 576 peptide action. We do not know whether NPY and αMSH release primarily occur from the

EXPERIMENTAL PROCEDURES 1119
Animals 1120 All animal care and experimental procedures were approved by the Institutional Animal Care and 1121 Use Committee at Beth Israel Deaconess Medical Center (BIDMC). Animals were housed in a 1122 12-hour-light/12-hour-darkness environment with standard mouse chow and water provided ad 1123 libitum, unless specified otherwise. Male and female mice older than 8 weeks were used in 1124 experiments, and the numbers of males and females were balanced to the degree allowed by 1125 each litter (see Table S1). Sex-specific analyses show consistent effects in males and females, 1126 but male mice were typically larger in size and ate more (Table S1). We used the following Experiments did not involve experimenter-blinding, but randomization was used to determine 1136 experimental order and group assignments.  concerns about potential impact on animal health, and the coordinates became the values below 1173 (which enables better views of PVH) once initial health concerns were found to be unsubstantiated. 1174 All AAVs were injected at a titer of 3-15 10 13 gc/ml (see below for volumes used). In experiments 1175 where unmixed cADDis and PDE4D3 viruses were used, both AAVs were pre-diluted 1:3 to a 1176 final titer of 3-4 10 13 gc/ml before injections to minimize potential effects on cell heath (which we 1177 verified in post hoc histological analyses). In the minority of cases where viral expression was 1178 absent (<10% of surgeries), we excluded the data from subsequent analyses. All animals were 1179 allowed to recover for at least 3 weeks prior to onsets of experiments. No obvious capsid 1180 competition was seen in histology. maximize the distance between cannula and GRIN lens, so as to avoid potential damage to the 1208 lens during tubing insertion and removal. A titanium head plate was centered over the GRIN lens 1209 and fixed to the skull using Metabond. A 3D-printed acrylic funnel was cemented onto the 1210 headplate to enable light-shielding during experiments. The area surrounding the lens was 1211 covered with Metabond and then dark dental cement to reduce autofluorescence. The GRIN lens 1212 was protected by a cut-off tip of an Eppendorf tube (Fisher) that was secured using Kwik-Cast 1213 (WPI). 1214 The experiments that involve two-photon fluorescence lifetime imaging of cADDis in the PVH 1215 during feeding used the same mice as those for optogenetic stimulation of POMC and AgRP 1216 axons.

Freely-moving 24-hr feeding, body-length, and body-weight measurements 1287
For freely-moving 24-hr feeding ( Figure 1E), body length ( Figure S1C), and body weight 1288 measurements ( Figure 1C), animals were singly housed, and body weight and 24-hr food intake 1289 of individual animals were measured every week following AAV injection. Body weight was 1290 measured on day 1 of each week. 24-hr food intake was measured for 3 consecutive days at the 1291 beginning of each week (days 1-3) and these values were averaged to account for daily variations 1292 in the food intake. 1293 For body length measurements, animals were maintained in group housing with their original 1294 littermates throughout the duration of the experiment. Body length (nose-to-anus length) was 1295 measured every week following AAV injection during brief isoflurane anesthesia. 1296 1297

Indirect calorimetry and MRI 1298
24-hour food intake (LabDiet 5008, 3.56 kCal/g), water intake, physical activity (beam breaks), 1299 oxygen consumption, carbon dioxide production, and body mass of single-housed mice were 1300 measured every 2 minutes using the Sable Systems Promethion indirect calorimeter in the BIDMC 1301 Energy Balance Core. Mice were weighed and body compositions were scanned using an 1302 EchoMRI 3-in-1 body composition analyzer (no anesthesia) before they were placed in the 1303 Promethion system for recording. All mice were allowed 12 hours of habituation in the system 1304 before recording the 72-hr experiment. From the measurements, energy intake, energy 1305 expenditure, respiratory exchange ratio, physical activity, and energy balance were calculated 1306 using (15 µl; e.g., Figure 6B) or 10 Ensure pulses (30 µl; e.g., Figure 6K) per trial. For all head-fixed 1317 feeding experiments, there was a 4-min waiting period before the first trial to record baseline 1318 sensor activity and bleaching rate. All dataset were collected as triplicates per condition. 1319 Food delivery during two-photon imaging was controlled with an Arduino 1320 (https://github.com/xzhang03/Train_generator). One session was performed per day. For two-1321 photon imaging experiments in Figures 6B-6G, 10 trials were conducted per session at a rate of 1322 1 trial per minute, and each trial used a 5-pulse train (300 ms between pulse onsets). 1323 Food delivery during photometry or behavioral experiments was controlled using the Nanosec 1324 photometry-behavioral system (https://github.com/xzhang03/NidaqGUI). In head-fixed binging 1325 assay, each lick triggers a pulse of Ensure, and we recorded the number of licks over an hour per 1326 mouse ( Figure 6I). Low-level biPAC stimulation was performed by pulsing 465 light (50 μW) at a 1327 pulse-width of 6 ms and a frequency of 50 Hz, with a resulting power of 15 μW. 1328 For photometry and head-fixed food intake experiments with trial structures (e.g., Figure 6J and 1329 7A), 50 trials were conducted per session at a rate of 1 trial per minute and each trial used a 10-1330 pulse train of Ensure deliveries (300 ms between Ensure pulse onsets, 3 μl/pulse, 30 μl total per 1331 trial). The increased number of trials and increased total Ensure delivery per trial during the 1332 photometry experiments were designed to partially satiate mice over the course of the experiment.

1333
For experiments using conditional food delivery (e.g., Figure 6J), a 2 kHz tone was played for 1 1334 second, during which mice must lick at least once to trigger the food delivery train. If triggered, 1335 the food delivery train started as soon as the tone was over. Otherwise, mice must wait until the 1336 next trial (1 trial/min), and the current trial was omitted in photometry analysis. At 10 pulses per 1337 train and 50 trains per session, the maximum amount of milkshake a mouse could consume there 1338 is 1.5 ml, which is roughly 50% of the absolute maximum volume that a mouse would consume 1339 in previous experiments 11 . For experiments involving biPAC stimulation (e.g., Figure 6J), a 1-s 1340 light pulse (465 nm, 1 mW peak, 70% duty cycle) was delivered 5 seconds before the tone in 50% 1341 of the trials (randomly selected, in Figure 6J). The 5-s delay was chosen to allow sufficient cAMP 1342 production before the tone (see Figure S5C). To verify satiation, we also sometimes (at least once 1343 per mouse per condition) gave the mouse the choice to perform an additional 10 trials 10 minutes 1344 after the regular experiments (see Figure 6K). 1345 For optogenetic experiments that also used i.c.v. infusion of neuropeptide agonists or antagonists, 1482 the drugs were pre-infused through the cannula 10 min before imaging started. For optogenetic 1483 experiments that also used i.p. injections (e.g., MTII), the injection was performed 15 min before 1484 imaging. 1485 For i.c.v. infusion experiments (e.g., Figure S2F) or i.p. injection experiments (e.g., Figure S5D) 1486 without optogenetic stimulation, we imaged for 15 min per session, and the drugs were introduced 1487 at 3 min into the recording. In these experiments, because cAMP changes are slow, FLIM was 1488 also used, and the FLIM data were acquired at 1 frame (5-sec acquisition) every 10 seconds 1489 throughout the session. 1490 For feeding experiments, simultaneous FLIM and non-FLIM imaging datasets (20 min per session) 1491 were acquired as described above. FLIM data were used to determine the overall changes in 1492 absolute cAMP levels, while non-FLIM (i.e. intensity-based imaging) data were used to visualize 1493 single-trial cAMP transients. FLIM data were acquired at 1 frame (5-sec duration) every 10 1494 seconds throughout the session. Non-FLIM imaging data were acquired at 15.5 fps and only 1 1495 FOV was imaged per session. Uncued food deliveries started at 4 min, and 10 trials were 1496 presented (1 min between trial onsets) using the methods described above. 1497 1498

Electrophysiology in acute slices 1499
To prepare ex vivo brain slices, 6-10 week-old mice were deeply anesthetized with isoflurane 1500 before decapitation and removal of the entire brain. Brains were immediately submerged in ice-1501 cold, carbogen-saturated (95% O2, 5% CO2) choline-based cutting solution consisting of (in mM): 1502 92 choline chloride, 10 HEPES, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 25 glucose, 10 MgSO4, 0.5 1503 CaCl2, 2 thiourea, 5 sodium ascorbate, 3 sodium pyruvate, oxygenated with 95% O2/5% CO2, 1504 measured osmolarity 310 -320 mOsm/L, pH= 7.4. Then, 275-300 μm-thick coronal sections were 1505 cut with a vibratome (Campden 7000smz-2) and incubated in oxygenated cutting solution at 34°C 1506 for 10 min. Next, slices were transferred to oxygenated ACSF (126 mM NaCl, 21.4 mM NaHCO3, 1507 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 10 mM glucose) at 34°C for an 1508 additional 15 min. Slices were then kept at room temperature (20-24°C) for 45 min until use. A 1509 single slice was placed in the recording chamber where it was continuously superfused at a rate 1510 of 3-4 mL per min with oxygenated ACSF. Neurons were visualized with an upright microscope 1511 (SliceScope Pro 1000, Scientifica) equipped with infrared-differential interference contrast and 1512 fluorescence optics. Devices, Axon Instruments) controlled the photostimulation output. 1525 Head-fixed photometry data were analyzed as described previously 55 1567 (https://github.com/xzhang03/Photometry_analysis). Pulses that trigger the photometry LED (50 1568 Hz, see above) were used to determine when the LED was on. For each pulse (6 ms), we took 1569 the median of the corresponding data points in the photodetector trace to quantify the photometry 1570 signal. This results in a 50 Hz trace which we then filtered with a 10 Hz low-pass filter. 1571 To estimate bleaching, we fitted the pre-stimulation data (first 4 min of each experiment) with a 1572 mono-exponential function and subtracted the estimated values from each timepoint in the 1573 photometry trace across the entire recording. Since all photometry experiments have a trial 1574 structure, we used the baseline fluorescence (e.g., the 10 s pre-stimulation window, see e.g., 1575 Figure S5A) to estimate ΔF/F0. The ΔF/F traces were then pooled across days and z-scored per 1576 mouse. To show single-trial photometry traces, we triggered photometry traces with cue onsets 1577 with a 10-s pre-cue baseline and a 50-s post-cue recording (see Figure S7A for an example 1578 experiment). For Figure 7A, we grouped the calcium transients in bins of 5 trials to show the 1579 gradual progression of excitation. 1580 1581

Pre-processing of two-photon imaging experiments 1582
Image registration was performed in MATLAB using a two-step process: 1) a rigid xy-translation 1583 step that is repeated 3 times (https://github.com/xzhang03/Tiff_preprocess), and 2) a non-rigid 1584 registration step using the Demonsreg function (https://github.com/xzhang03/Demonsreg-1585 Oneshot). For experiments that involve simultaneous imaging of multiple z-planes, each z-plane 1586 was registered independently. To extract cell bodies, we down-sampled images spatially by a 1587 factor of two. Then, we used Cellpose 2.0 96 to segment soma ROIs. Repeating somas from 1588 multiple FOVs were excluded during analysis. For fixed-slide imaging, the number of ROIs was 1589 reported as the number of somas expressing the markers. We note that this number likely only 1590 reflects 50% of total cell counts, as we imaged every other slice. 1591 To extract neurite ROIs, we retrained three built-in Cellpose modelscyto, nuclei, and the base 1592 networkto manually labeled neurites in three steps on an NVIDIA RTX3080 GPU: 1) 100 1593 iterations of a 31-image set, 2) 500 additional iterations of the same set, and 3), 2000 iterations 1594 of a 119-image set. We eventually decided to use the nuclei-based neurite model for subsequent 1595 analyses. Training progress, segmentation results, and the models are all deposited to a public 1596 repository (https://github.com/xzhang03/cellpose_GRIN_dendrite_models). After segmenting 1597 neurites, neurite ROIs were then manually matched to somas of the same field of view for future 1598 analysis. 1599 For cross-day experiments (e.g., Figures 6B-6G), soma-ROIs in the same FOV were also 1600 matched manually. 1601 After segmentation, we calculated the fluorescence traces from soma and neurite ROIs, and 1602 subtracted from them the fluorescence traces of the surrounding neuropil rings. Neuropil rings 1603 were calculated by first dilating the ROI by 14 pixels and subtracting all ROIs from the dilated 1604 mask. If the resulting neuropil ring has fewer than 2500 pixels, the process is repeated with 1605 incrementing dilating size. Mean neuropil traces were subtracted from the mean ROI trace at 1:1 1606 scaling. The underlying assumption for the no-scaling subtraction is that if a given pixel contains 1607 photons from both the intended ROI (e.g., soma) and the neuropil, the neuropil photon 1608 contribution should be similar at the soma and the surrounding area. Therefore, the subtraction 1609 was done to remove global influences from the estimation of neurons' responses to stimulation. 1610 The traces were then triggered relative to the onset of optogenetic stimulation or food delivery, 1611 and the relative changes (ΔF/F0) were calculated. We used a 20-s pre-stimulation and 110-s post-1612 stimulation window to display most cAMP traces (e.g., Figure 2I). The 110-s post-stimulation estimation of the chance level of correlation needs to be calculated on a case-by-case basis, 1703 which we did through bootstrapping. We also verified the main conclusions with an independent 1704 method, F1 score 155 (also referred to as Dice correlation), as well as Pearson correlation of ΔF/F0 1705 values. 1706 For analyses that also involve distance between ROIs, we used inter-centroid distances and 1707 adjust for GRIN lens magnification (2.6x magnification for doublets) as needed for in vivo

Code availability 1745
The custom analysis codes and designs in this paper are publicly available on Github 1746 (https://github.com/xzhang03/Code-for-Zhang-and-Kim-et-al/). n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001 for all figures. See Table S1 for statistical details  1768 and sex-specific characterizations.    where they compete to control cAMP in PVH MC4R neurons (B). Feeding reduces AgRP neuron activity 1924 while elevating POMC neuron activity, a combination that results in sustained cAMP elevation in PVH MC4R 1925 neurons for tens of minutes (C). In turn, elevated cAMP gradually potentiates feeding-related excitatory 1926 inputs to satiety-promoting PVH MC4R neurons (D), thereby promoting satiation (E). 1927 1928